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. Author manuscript; available in PMC: 2016 Jun 14.
Published in final edited form as: Nat Cell Biol. 2015 Dec 14;18(2):202–212. doi: 10.1038/ncb3289

Figure 1. Rpt3-T25 is dynamically phosphorylated during cell cycle.

Figure 1

(a) Validation of anti-pT25 phospho-specific antibody. 293T cells transfected with a vector control (−) or HA-Rpt3 (WT or T25V) were subjected to anti-HA immunoprecipitation (IP). Samples were then treated with or without λ-phosphatase and analyzed by western blot.

(b) In vivo phosphorylation of Rpt3-T25. Whole brain from E12.5 mouse embryos was homogenized and subjected to immunoprecipitation (IP) with normal IgG or anti-Rpt3 antibody. T25 phosphorylation was determined by western blot.

(c) Phospho-T25 was detected from the purified 26S proteasome. Lysates from 293T cells stably expressing Rpn11-HTBH (for proteasome purification) or the HTBH tag only (“−”, negative control) were subjected to streptavidin pulldown. T25 phosphorylation was determined by western blot.

(d) Reversible phosphorylation of Rpt3-T25. 293T Rpn11-TBHA cells were either untreated (−) or pretreated with 25 nM Calyculin A for 10 min before harvest. The 26S proteasome was purified and pT25 was determined by western blot.

(e) HaCaT cells were either untreated (“+” serum) or serum-starved (“−”) for 48 hours. Cell lysates were probed with the indicated antibodies.

(f) Contact inhibition reduces Rpt3-T25 phosphorylation. WT MEFs expressing Rpn11-TBHA were grown to 100% confluence and contact-inhibited (C. I.) in the presence of serum for 48 hrs. Half of the cells were frozen immediately (in G1 phase) while the other half were allowed to resume growth at a lower density for 18 hrs. The proteasomes were purified from both samples at the same time and phospho-T25 was probed.

(g) Cell cycle-regulated Rpt3-T25 phosphorylation. HaCaT Rpn11-TBHA cells enriched in M phase with nocodazole (Ndz) were collected by mitotic shake-off and released. At the indicated time points. proteasomes from each sample were affinity-purified by streptavidin pulldown, and phospho-T25 and proteasome subunits were probed. Cell cycle proteins from whole cell lysate (WCL) were also probed to show progression along cell cycle.